1. Volume 43, Issue 5, Pages 703-712 (September 2011)
Interaction of Arabidopsis DET1 with CCA1 and LHY in Mediating Transcriptional Repression in the Plant Circadian Clock 
On Sun Lau, Xi Huang, Jean-Benoit Charron, Jae-Hoon Lee, Gang Li, Xing Wang Deng 
Molecular Cell 
Volume 43, Issue 5, Pages (September 2011)
DOI: /j.molcel
Copyright © 2011 Elsevier Inc. Terms and Conditions

2. Molecular Cell 2011 43, 703-712DOI: (10.1016/j.molcel.2011.07.013)
Copyright © 2011 Elsevier Inc. Terms and Conditions

3. Figure 1
DET1, a Component of the CDD Complex, Directs Transcriptional Repression
(A and B) Transcriptional repression assays in yeast with a lacZ reporter driven by basal promoters with either Gal4 operators (Gal4op) (SS38 in A), LexA operators (LexAop) (JK1621 in B), or no operator (pLGΔ312S in B) upstream. In (A), the reporter was integrated into the yeast genome and plasmids encoding Gal4-DNA binding domain (Gal4DBD) alone (negative control) and Gal4DBD fused with DET1 or LUG (positive control) were independently transformed into yeast. β-galactosidase activities with the different effectors are shown (mean ± SD; n ≥ 6). In (B), the two reporters were cotransformed with different LexA-fusion proteins. pLGΔ312S is a control reporter for detecting potential effector's nonspecific activity, while LexA alone acts as a negative control. Reporter activities were measured as in (A).
(C) Transient transcriptional repression assays in plants with a luciferase (LUC) reporter driven by a minimal CaMV 35S promoter (m35Spro) with or without Gal4ops upstream. The reporter vectors also contain a 35S-driven Renilla luciferase (REN) for signal normalization. The reporters were cotransformed independently with a plasmid containing either the GAL4DBD alone or GAL4DBD-DET1 into the leaves of Nicotiana benthamiana. Activities of firefly luciferase (LUC) normalized to Renilla luciferase (REN) are shown (mean ± SD; n ≥ 4). Activities with the control effectors: Gal4DBD alone (A and C) or LexA alone (B), are set to 1.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2
DET1 Interacts with Two MYB Transcription Factors, CCA1 and LHY, which Are Core Components of the Plant Circadian Clock
(A) In vitro GST pull-down assay between recombinant His-DET1 and GST-fused HY5, HFR1, and CCA1. His-DET1 was incubated with immobilized GST or the GST-fused proteins, and immunoprecipitated fractions were probed with an anti-DET1 antibody.
(B) Liquid yeast two-hybrid assays for interaction between CCA1 and deletion series of DET1. Left panel shows the constructs encoding LexA alone (control) and LexA-fused DET1 and its fragments. Right panel shows the corresponding β-galactosidase activities with either Activation Domain (AD) alone or AD-CCA1 (mean ± SD; n ≥ 4).
(C) In vitro GST pull-down assay between His-T7-tagged CCA1 or LHY and a GST-fused N-terminal fragment of DET1 (26–87 aa). The His-T7-tagged proteins were incubated with immobilized GST or the GST-fused DET1 fragment, and immunoprecipitated fractions were probed with an anti-T7 and anti-GST antibody. IB: immunoblot.
(D) Bimolecular fluorescence complementation (BiFC) analysis for in vivo interaction between DET1 and CCA1. N- and C-terminal fragments of yellow fluorescence protein (YFP; YFPN and YFPC) were fused to DET1 and CCA1, respectively. Combinations of their encoding plasmids and controls (indicated on the left panel) were transiently transformed into leaves of Nicotiana benthamiana. Presence of YFP signal (YFP) indicates reconstitution of YFP through protein interaction of the tested pairs. Positions of the nuclei were stained with DAPI and marked by arrows.
(E and F) Firefly luciferase complementation imaging (LCI) assays for in vivo interaction between DET1, CCA1 and LHY. CCA1, LHY, and an N-terminal fragment of CCA1 (1–338 aa) were fused with the N-terminus of luciferase (LUCn), and DET1 was fused with the C-terminal part (LUCc). Plasmids were transformed into plants as in (D) and LUC activities above background indicate LUC reconstitution and interaction. Relative LUC activities (luminescence intensity/leaf area) of the indicated transformation (lower panel) are shown (mean ± SD; n ≥ 4).
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3
DET1 Binds to Target Genes of CCA1 and LHY and Regulates Their Circadian Expression
(A) ChIP-quantitative PCR (ChIP-qPCR) analyses of DET1 binding on TOC1 promoter. ChIP assays were performed on seedlings of WT (Col) collected at zeitgeber time (ZT) 0 and 12 and the cca1-11 lhy-21 double mutant collected at ZT 0, with and without an anti-DET1 antibody. ChIP DNA was quantified by quantitative real-time PCR with primers and probes specific to the evening element (EE)-containing region of TOC1 promoter (TOC1 Pro) and the 3′ untranslated region of TOC1 (TOC1 3′ UTR) (control). Signals were normalized to the input DNA (mean ± SD; n ≥ 3). Ab: antibody.
(B) Transient transcriptional repression assays in plants for DET1 effect on an EE-containing reporter (2xEE) and a TOC1 promoter-driven reporter (TOC1 Pro). Assays were carried out as in Figure 1C. Relative reporter activities (LUC/REN) with the indicated effectors are shown (mean ± SD; n ≥ 4). Activities of reporters with the control effector, Gal4DBD, are set to 1.
(C and D) Circadian rhythms of TOC1 (C) and GI (D) expression in WT (Col) and det1-1 under continuous light (LL). Plants were grown with long day cycles (16L8D) for 8 days and then released to LL. RNA levels were quantified by TaqMan real-time RT-PCR, and signals were normalized to 18S (mean ± SD; two biological and two technical replicates).
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4 DET1 Is Important for CCA1 Function
(A and B) Circadian rhythms of TOC1 (A) and GI (B) expression in CCA1-OX and CCA1-OX;det1-1 under continuous light (LL). Experiments were performed as in Figures 3C and 3D.
(C) Nine-day-old seedlings of WT (Col), CCA1-OX, CCA1-OX;det1-1, and det1-1 grown under long day (LD; 16L8D). Hypocotyl length measurements of the seedlings are shown (mean ± SD; n ≥ 20).
(D) Five-week-old plants of CCA1-OX, CCA1-OX;det1-1, det1-1, and Col grown under LD. The number of rosette leaves at the time of flowering is shown (mean ± SD; n ≥ 12).
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5
A Model for the Functional Interaction between the CDD Complex with CCA1 and LHY in Regulating the Central Loop of the Arabidopsis Circadian Clock
At dawn, the morning-expressed CCA1 and LHY physically interact with DET1, likely in the form of the CDD complex, and recruit it to their evening-phased gene targets, such as TOC1. The CDD complex then exerts its repression activity on these CCA1/LHY targets and suppresses their expression. Gradual reduction in the level of CCA1 and LHY throughout the day reduces the association of the CDD complex on the genes and allows accumulation of TOC1 transcripts, which peak at dusk. TOC1 in turn induces expression of CCA1 and LHY, where their proteins reach their peak at dawn, and the cycle continues. CHE, a CCA1-binding transcription factor, forms a reciprocal negative loop with CCA1. TOC1 can directly interact with CHE and antagonize its repression on CCA1.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2011 Elsevier Inc. Terms and Conditions